One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester.

Bastings MM, van Baal I, Meijer EW, Merkx M - BMC Biotechnol. (2008)

Bottom Line:
Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.An efficient method was developed for the production of disulfide bond containing proteins with C-terminal thioesters.Introduction of a MESNA/diMESNA redox couple resulted in simultaneous on-column refolding, purification and thioester generation of the model protein Ribonuclease A.

Background: Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.

Results: A novel redox buffer consisting of MESNA and diMESNA showed a refolding efficiency comparable to that of GSH/GSSG and prevented loss of the protein's thioester functionality. Moreover, introduction of the MESNA/diMESNA redox couple in the cleavage buffer allowed simultaneous on-column refolding of Ribonuclease A and intein-mediated cleavage to yield Ribonuclease A with a C-terminal MESNA-thioester. The C-terminal thioester was shown to be active in native chemical ligation.

Conclusion: An efficient method was developed for the production of disulfide bond containing proteins with C-terminal thioesters. Introduction of a MESNA/diMESNA redox couple resulted in simultaneous on-column refolding, purification and thioester generation of the model protein Ribonuclease A.

Mentions:
The glutathione redox couple is the most common redox couple used to assist in protein refolding. To investigate the stability of protein thioesters in a glutathione redox buffer, green fluorescent protein (GFP) with C-terminal MESNA thioester was used as a test protein and incubated for 10 hours in glutathione refolding buffer. ESI-MS analysis showed significant transthioesterification and subsequent hydrolysis of the glutathione thioester (Figure 1a). To avoid the problem of transthioesterification with components of the redox buffer present in refolding buffers, we developed a MESNA-based redox buffer by synthesizing the oxidized, disulfide form of MESNA, 2,2-dithiobis(ethanesulfonate) (diMESNA) (Figure 2) [20]. Figure 1b shows that GFP-MESNA is stable in a MESNA-based redox buffer. A small peak corresponding to hydrolyzed GFP was observed after 10 h incubation at RT, but the amount of hydrolysis is significantly reduced compared to the incubation with glutathione redox buffer. The efficiency of the MESNA/diMESNA redox couple in protein refolding was tested using bovine pancreatic Ribonuclease A (RNase A). RNase A is a small protein of 124 amino acids that contains 4 disulfide bridges [21] and has been extensively used as a model to study the effects of disulfide bonds on protein refolding. Commercially available RNase A, purified by HPLC was unfolded in a 6 M guanidinium hydrochloride (Gu·HCl) denaturating buffer using tris(2-carboxyethyl)phosphine (TCEP) as reducing agent. Refolding was achieved by dilution of the unfolded RNase A in 3/1 mM MESNA/diMESNA containing refolding buffer. To compare this refolding buffer with conventional refolding conditions, refolding was also performed in a refolding buffer containing 3/1 mM GSH/GSSG. Refolding efficiencies were calculated based on an activity assay using the fluorescent 6-FAM-dArUdAdA-6-TAMRA substrate for RNase [22]. Kinetic measurements of the increase in FAM emission at 515 nm upon substrate cleavage by RNase A were performed. A linear fit to the initial 30 seconds of each activity assay yielded the enzyme's initial velocity. A calibration curve using known concentrations of commercial RNase A was used to determine the concentration of enzymatically active RNase A after refolding and thus the refolding efficiency (Figure 3a). Using this activity assay, refolding efficiencies of 58 ± 5% for the MESNA couple compared to 72 ± 5% of the glutathione couple after 24 hours of refolding were determined. These results show that the MESNA/diMESNA redox couple is a suitable alternative redox buffer for the refolding of RNase A (Figure 3b).

Mentions:
The glutathione redox couple is the most common redox couple used to assist in protein refolding. To investigate the stability of protein thioesters in a glutathione redox buffer, green fluorescent protein (GFP) with C-terminal MESNA thioester was used as a test protein and incubated for 10 hours in glutathione refolding buffer. ESI-MS analysis showed significant transthioesterification and subsequent hydrolysis of the glutathione thioester (Figure 1a). To avoid the problem of transthioesterification with components of the redox buffer present in refolding buffers, we developed a MESNA-based redox buffer by synthesizing the oxidized, disulfide form of MESNA, 2,2-dithiobis(ethanesulfonate) (diMESNA) (Figure 2) [20]. Figure 1b shows that GFP-MESNA is stable in a MESNA-based redox buffer. A small peak corresponding to hydrolyzed GFP was observed after 10 h incubation at RT, but the amount of hydrolysis is significantly reduced compared to the incubation with glutathione redox buffer. The efficiency of the MESNA/diMESNA redox couple in protein refolding was tested using bovine pancreatic Ribonuclease A (RNase A). RNase A is a small protein of 124 amino acids that contains 4 disulfide bridges [21] and has been extensively used as a model to study the effects of disulfide bonds on protein refolding. Commercially available RNase A, purified by HPLC was unfolded in a 6 M guanidinium hydrochloride (Gu·HCl) denaturating buffer using tris(2-carboxyethyl)phosphine (TCEP) as reducing agent. Refolding was achieved by dilution of the unfolded RNase A in 3/1 mM MESNA/diMESNA containing refolding buffer. To compare this refolding buffer with conventional refolding conditions, refolding was also performed in a refolding buffer containing 3/1 mM GSH/GSSG. Refolding efficiencies were calculated based on an activity assay using the fluorescent 6-FAM-dArUdAdA-6-TAMRA substrate for RNase [22]. Kinetic measurements of the increase in FAM emission at 515 nm upon substrate cleavage by RNase A were performed. A linear fit to the initial 30 seconds of each activity assay yielded the enzyme's initial velocity. A calibration curve using known concentrations of commercial RNase A was used to determine the concentration of enzymatically active RNase A after refolding and thus the refolding efficiency (Figure 3a). Using this activity assay, refolding efficiencies of 58 ± 5% for the MESNA couple compared to 72 ± 5% of the glutathione couple after 24 hours of refolding were determined. These results show that the MESNA/diMESNA redox couple is a suitable alternative redox buffer for the refolding of RNase A (Figure 3b).

Bottom Line:
Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.An efficient method was developed for the production of disulfide bond containing proteins with C-terminal thioesters.Introduction of a MESNA/diMESNA redox couple resulted in simultaneous on-column refolding, purification and thioester generation of the model protein Ribonuclease A.

Background: Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.

Results: A novel redox buffer consisting of MESNA and diMESNA showed a refolding efficiency comparable to that of GSH/GSSG and prevented loss of the protein's thioester functionality. Moreover, introduction of the MESNA/diMESNA redox couple in the cleavage buffer allowed simultaneous on-column refolding of Ribonuclease A and intein-mediated cleavage to yield Ribonuclease A with a C-terminal MESNA-thioester. The C-terminal thioester was shown to be active in native chemical ligation.

Conclusion: An efficient method was developed for the production of disulfide bond containing proteins with C-terminal thioesters. Introduction of a MESNA/diMESNA redox couple resulted in simultaneous on-column refolding, purification and thioester generation of the model protein Ribonuclease A.